The loss of osteochondral tissue from the joints following extensive arthritis, necrosis or as a result of a trauma or the removal of a tumour requires reconstructive surgery to repair the defect.
The treatment of such defects by means of joint replacements is limited by the fact that the non-biological materials used are prone to wear and tear, and that the replacements themselves may be difficult to move.
Alternatively, it is possible to resort to autologous, allogeneic or xenogeneic bone grafts. The ideal way of substituting bone is by grafting autologous tissue, which has a better osteogenic capacity than allografts and xenografts, as shown by the presence, during its resorption, of mesenchymal cells that differentiate into osteogenic and chondrogenic cell lines (K. L. B. Brown et al., Surg. 1982, 64A, 270-279). Moreover, allografts and xenografts carry antigens of histocompatibility, making it necessary to administer immunosuppressive treatment to the patient, while autografts do not trigger any immunological response. Besides this, there is the possibility that allografts and xenografts may transmit viruses to the recipient, such as HIV, hepatitis or BSE.
Since bone autografts cause a second trauma to the patient when the graft is taken from the donor site, and because of the paucity of available tissue, the use of this procedure is rather limited.
Orthopaedic research has long been focused on the study of new, artificial materials suitable for osteochondral grafts which would reduce or eliminate the need to resort to tissue grafts.
In order to meet requirements, a material must be biocompatible, bioresorbable at a rate that is comparable to bone growth, able to bear load, be easy to sterilise and process, be osteoactive, that is, it must be an osteoinductor, to induce mesenchymal cell differentiation into bone progenitor cells, and an osteoconductor, to enable the growth of bone within the graft.
The techniques reported in the literature for bone regeneration refer to the use of ceramics, polymers, composite materials and bioactive molecules.
The fact that, from a chemical and structural point of view, calcium phosphates are similar to the mineral part of bone that is mainly constituted by biological hydroxyapatites, has promoted the use of ceramics as biomaterials to induce osteogenesis.
The ceramics most commonly used are beta-tricalcium phosphate (TCP) and synthetic hydroxyapatite. They have proved to be osteoconductors, thanks to their porosity that favours cell colonisation and bone growth.
Studies conducted with subcutaneous implants in syngeneic rats have shown that the combination of bone marrow cells and porous ceramics promote osteogenesis, with the formation of new bone within the pores. Moreover, small, isolated areas of cartilage without any appreciable endochondral ossification have been observed (H. Ohgushi et al., J. Orthop. Res., 1989, 4, 568-578).
The widespread use of polymers is explained by the possibility of obtaining different compositions and structures able to satisfy the requirements of the specific applications as well as the property of biodegradation. It is known that polylactic acid, polyglycolic acid and copolymers or derivatives thereof can be used, in various forms, as biomaterials for the growth of osteocytes. The main disadvantage of using such scaffolds is represented by the immune response directed against the implanted material.
In order to create a biomaterial that is satisfactory both from a mechanical and biological point of view, various composite materials formed by mixtures of polymers and calcium phosphates have been investigated.
It is also known that scaffolds containing at least one hyaluronic acid derivative can be used as biomaterials for tissue growth.
Hyaluronic acid is a polysaccharide ether composed of alternate residues of D-glucuronic acid and N-acetyl-glucosamine. It is a linear polymer chain with a molecular weight that varies between 50,000 and 13,000,000 Da, depending on its source and on the methods of preparation and determination that are used. It is present in nature in the pericellular gels, in the fundamental substance of the connective tissue of vertebrate organisms of which it is the main component, in the synovial fluid of joints, in the vitreous humor, in human umbilical cord and in rooster combs.
Hyaluronic acid plays a vital role in many biological processes such as hydration, proteoglycan organisation, cell differentiation, proliferation and angiogenesis (J. Aiger et al., L. Biomed. Mater. Res. 1998, 42, 172-181).
It is also known that hyaluronic acid fractions can be used to enhance tissue repair, to substitute the intraocular fluid, or they can be administered by the intra-articular route to treat joint pathologies, as described in European patents No.s 0138572 and 0535200.
Hyaluronic acid plays a fundamental role in the tissue repair process, especially in the early granulation stage, stabilising the coagulation matrix and controlling its degradation, favouring the recruitment of cells involved in the inflammatory process, such as fibroblasts and endothelial cells and, lastly, orienting the subsequent migration of epithelial cells.
It is known that the application of hyaluronic acid solutions can accelerate the tissue repair process in patients with wounds or burns. The role of hyaluronic acid in the various phases of the tissue repair process has been described by the construction of a theoretical model by P. H. Weigel et al., J. Theor. Biol., 119:219, 1986.
The use of low-molecular-weight fractions of hyaluronic acid and the autocrosslinked derivatives thereof is also known in the preparation of pharmaceutical compounds that are osteoinductors (WO 93/20827).
The total or partial esters of hyaluronic acid (HYAFF®) and its autocrosslinked derivatives (ACP®) are known, as is their use in the pharmaceutical and cosmetic fields and in that of biodegradable materials (U.S. Pat. Nos. 4,851,521; 4,965,353 and 5,676,964).
In particular, patent application No. WO 93/20858 describes binding solutions and pastes containing hyaluronic acid and/or the ester derivatives thereof used as bone fillers in surgery.
Lastly, esters of hyaluronic acid have been processed in the form of non-woven structures according to the process described in U.S. Pat. No. 5,520,916.
Hyaluronic acid derivatives in three-dimensional form and, in particular, partial and total esters of hyaluronic acid (HYAFF®) processed in the form of non-woven tissues have been used as scaffolds in the preparation of biological materials containing cells and/or products generated from such cells.
For example, it has recently investigated the possibility of using the benzyl ester of hyaluronic acid (HYAFF®-11) as a scaffold, in a form of a non-woven fiber structure, for the culture of human chondrocytes in tissue-engineering procedures of cartilage reconstruction. In these 3D cultures, chondrocytes were able to produce hyaline cartilage-specific matrix molecules like collagen type II or proteoglycans, (J. Aiger et al., L. Biomed. Mater. Res. 1998, 42, 172-181).
We can, moreover, mention patent application No. WO 97/18842 that describes a material containing:                a. an efficient culture of autologous or homologous stem cells from bone marrow, partially or completely differentiated into cells of a specific connective tissue, containing moreover the matrix secreted by said cells, or alternatively,        a′. the extra-cellular matrix secreted by completely or partially differentiated bone marrow stem cells or, alternatively, by mature cells of the tissue;        b. a three-dimensional matrix consisting of hyaluronic acid derivatives and, in particular, partial or total esters (HYAFF®).        
Hyaluronic acid derivatives in the form of sponges (HYAFF®-11 sponge, made of benzyl ester of hyaluronic acid, and ACP® sponge, made of cross-linked hyaluronic acid) used in combination with mesenchymal cells, implanted subcutaneously in nude mice, have exhibited a better osteogenic and chondrogenic capacity in terms of quantity of tissue formed, than porous ceramics (L. A. Solchaga et al., J. Orthop. Res., 17, 1999, 205-213).
In another experiment, the above said two hyaluronan derivatives-based biomaterials were tested for their ability to enhance the natural healing response of the articular cartilage for self-repair. The introduction of these polymers into osteochondral defects (made on the femoral condyles of rabbits) provides an appropriate scaffolding for the reparative process: in fact, the defects treated with HYAFF®-11 and ACP® sponges exhibited good bone fill and the surface of the condyles was mainly constituted of hyaline cartilage. (L. A. Solchagaet al., J. Orthop. Res., 18, 2000, 773-780).
It has proved extremely important to guarantee the development of a stable interface between the joint cartilage and underlying bone structure when repairing osteochondral defects.
Composite materials constituted by engineered cartilage stitched over an osteoconductor biomaterial scaffold, for the regeneration of osteochondral tissue have been implanted in defects created in rabbit joints. Six months later, it was possible to observe the remodelling of the composite material into an osteochondral tissue structurally similar to the natural variety, with a clear tidemark between the cartilage and the subchondral bone (D. Schaefer et al., 4th Annual Meeting of the Orthopaedic Research Society, 2000).
Therefore, although the use of hyaluronic acid derivatives and ceramic materials was already known both for the regeneration/repair of cartilage and bone and as scaffolds for the culture of differentiated and non-differentiated cells, and that an expert in the field could, consequently, have deduced that it was possible to use either of these materials to make osteochondral grafts, it could not have been foreseen, as demonstrated by the present invention, that once the two materials had been coupled, it would be possible to obtain separate formations of cartilage and bone that are structurally integrated but with no penetration between the tissues, like natural osteochondral tissue.